The invention was supported in part by grant number CDR-88-03014 from the National Science Foundation. The U.S. government has rights in the invention.
This invention relates to processes for purifying a biologically active ligate. An example of such a process includes affinity chromotography.
Affinity chromatography is a procedure commonly used for purifying a biologically active ligate, for example, a protein, such as an antibody or antigen, or a nucleic acid. Generally, in this procedure a ligand capable of specifically binding to the ligate is bound to a solid phase, such as a glass bead or nitrocellulose membrane. The ligand is bound in a manner which allows the specific binding of the ligate to the ligand. The ligate is then contacted with the bound ligand under conditions in which a ligand/ligate complex is formed on the solid phase, and the solid phase is washed to remove contaminating substances. During washing the complex remains bound to the solid phase. Subsequently, the ligate is caused to separate from the ligand, and thus the solid phase, by provision of an eluting solution. The ligate is then recovered from the eluting solution.
This procedure exploits the specificity of biological interaction between a ligand and a ligate, and is a highly specific method for isolating a biological material. Only a ligate having a sufficient affinity for the ligand on the solid support is retained on that solid support. Other compounds within the sample containing the ligate are not bound to the ligand, and are washed from the solid support during the procedure.
A suitable eluting solution is chosen from a variety of buffers, including solutions containing high concentrations of an acid, a base, a chaotropic salt, or a denaturing agent. This solution causes the ligand/ligate complex to become unstable, and thus release of the ligate into the eluting solution.